Anal. Chem. 2004, 76, 110-113
Kinetics of Metal-Fulvic Acid Complexation Using a Stopped-Flow Technique and Three-Dimensional Excitation Emission Fluorescence Spectrophotometer F. C. Wu,*,† R. B. Mills,† R. D. Evans,† and P. J. Dillon†,‡
Environmental and Resource Studies, Department of Chemistry, Trent University, Peterborough, Ontario K9J 7B8, Canada
A stopped-flow technique and three-dimensional excitation emission (Ex/Em) fluorescence spectrophotometer were used to detect the full fluorescence spectral kinetic changes that occurred during the complexation between fulvic acid (FA) and several metals [Cu(II), Ni(II), Co(II), Cd(II) and Ca(II)]. The study was carried out with a fulvic acid isolated from Cavan Bog, Canada. At pH 7, the FA reacted rapidly with all metals studied. Two major kinetically distinguishable binding sites on FA (“fast” and “slow”), having reaction half-lives of 1.3-3.9 and 34.769.3 s, respectively, were identified using pseudo-firstorder kinetic plots. Kinetic changes of Ex and Em wavelengths of the fluorescence maximums also indicate two major binding sites. For the fast-reacting binding site, the rate constant and the site relative contribution were as follows, Cu2+ > Ni2+ > Co2+ > Cd2+ > Ca2+, which agrees with the Irving-Williams series, indicating that complexation kinetics are affinity dependent. Within each kinetic phase, both Ex and Em wavelengths of fluorescence maximums increased with time, indicating the occurrence of structural changes during the binding process. Based on the results obtained, the use of full fluorescence spectra appears to be a promising tool for further understanding metal-FA complexation mechanisms. The binding of metal ions to dissolved organic matter (DOM) is of great interest because of its importance in regulating metal speciation, toxicity, bioavailability and transport in aquatic environments.1,2 A major fraction of DOM in most waters is humic substances (HS), which can be operationally separated into fulvic acid (FA), humic acid, and humin. In terms of metal binding capacity in natural waters, HS are the most important organic ligands.1-3 In the past decades, various methods, e.g., ion-selective electrodes, fluorescence spectroscopy, electron spin resonance, thermal lensing, and cathodic stripping voltammetry, have been * To whom correspondence should be addressed: (e-mail)
[email protected]; (phone) (705) 748-1011, ext 1370; (fax) (705) 748-1569. † Environmental and Resource Studies. ‡ Department of Chemistry. (1) Breault, R.; Colman, J.; Akien, G.; McKnight, D. Environ. Sci. Technol. 1996, 30, 3477-3480. (2) Xue, H.; Oestreich, A.; Kistler, D.; Sigg, A. Aquat. Sci. 1996, 58, 69-87. (3) Petersson, C.; Bishop, K.; Lee, Y.; Lee, B. Water, Soil Air Pollut. 1995, 80, 971-985.
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used to study the complexation between metals and DOM. Models have been developed, and stability constants and ligand concentrations in natural waters have been reported and reviewed.4-6 Although thermodynamic constants are very useful, kinetic studies are badly needed to better interpret the metal-DOM binding mechanism.7-10 Fluorescence spectroscopy offers a unique perspective on metal-DOM binding kinetic studies as it directly observes the DOM ligand and is fast enough to detect kinetic changes at a time scale of a few seconds. In the limited reports available on kinetic studies,7-10 Ex or Em spectra have been used to study the kinetics of fluorescence quenching or enhancement when DOM or HS binds with metals, e.g., Cu, Al, and Ni. Although major changes in total fluorescence were observed during the complexation,7-10 these studies reported only fluorescence intensities at specific Ex/Em wavelengths; thus, it is difficult to associate the changes with specific structures or environmental changes that may occur during the binding process. By using a highly sensitive and fast three-dimensional Ex/Em fluorescence matrix (3DEEM) spectrophotometer, we can observe the full Ex and Em fluorescence spectral changes that occur during the complexation between metals and DOM at a time scale of a few seconds. 3DEEM spectroscopy has been used successfully to study the chemical structures of HS and DOM due to the wealth of information it provides including patterns of maximum fluorescence intensity, fluorescence intensity, and Rayleigh scattering.11-14 Thus, the 3DEEM spectrophotometer coupled with a stoppedflow technique provides qualitative structural information in addition to fluorescence intensity versus time data, pertaining to the metal and FA complexation process. (4) Donat, J. R.; Bruland, K. W. In Trace Elements in Natural Waters; Salbu, B., Steinnes, E., Eds.; CRC Press: Boca Ration, FL., 1995; p 302. (5) Tanoue, E.; Midorikawa, T. In Biogeochemical Processes and Ocean Flux in the Western Pacific; Saki, H., Nazaki, Y., Eds.; Terra Scientific Publishing: Tokyo, 1995; p 201. (6) Town, R. M.; Filella, M. Aquat. Sci. 2000, 62, 252-295. (7) Mak, M. K. S.; Langford, C. H. Can. J. Chem. 1982, 60, 2023-2028. (8) Plankey, B. J.; Patterson, H. H. Environ. Sci. Technol. 1987, 21, 595-602. (9) Hering, J. G.; Morel, F. M. M. Environ. Sci. Technol. 1990, 24, 242-252. (10) Lin, C. F.; Lee, D. Y.; Chen, W. T.; Lo, K. S. Environ. Pollut. 1995, 87, 181-187. (11) Coble, P. G.; Green, S. A.; Blough, N. V.; Gagosian, R. B. Nature 1990, 348, 432-435. (12) Mopper, K.; Schultz, C. A. Mar. Chem. 1993, 41, 229-240. (13) Wu, F. C.; Tanoue, E. Environ. Sci. Technol. 2002, 35, 3646-3652. (14) Ryan, D. K.; Weber, J. H. Anal. Chem. 1982, 54, 986-990. 10.1021/ac030005p CCC: $27.50
© 2004 American Chemical Society Published on Web 11/22/2003
Figure 1. Scheme of the stopped-flow technique and 3DEEM spectrophotometer.
EXPERIMENTAL SECTION The humic material used in this work was fulvic acid isolated from Cavan Bog, ON, Canada, in August 1997 (CBFA). The isolation procedure followed the methods for XAD-8 extraction as published by Thurman and Malcolm.15 Exceptions to the method include the use of 0.45-µm cellulose acetate membranes in place of silver filter membranes and the air-drying of samples in place of freeze-drying. CBFA has been well characterized and described elsewhere.16,17 All chemicals used were reagent grade. The buffer was 0.01 M borate, pH was adjusted to 7.0 with HCl or NaOH, and NaCl was used to adjust the ionic strength to 0.1 M. Metal salts used were Cd(NO3)2‚4H2O, Co(NO3)2‚6H2O, CaCl2‚2H2O, Ni(NO3)2, and CuSO4‚5H2O. Solutions containing 50 mg L-1 CBFA and 1 × 10-4 M of metal ion were prepared for the kinetic study unless stated otherwise. Kinetic runs were performed with a stopped-flow apparatus designed in the laboratory (Figure 1). Buffered solutions of CBFA and metal salts were delivered by a peristaltic pump into a mixing chamber and then directly into a 25-µL quartz flow cell of the spectrophotometer for observation. This kinetic apparatus allows the two solutions to mix in less than 150 ms. The 3DEEM spectrophotometer used (SPEX, Jobin Yvon, Instruments S.A. Inc.) was equipped with a 150-W xenon short arc lamp and a multichannel charge-coupled device detector. It can be optimized to allow for a full 300 nm × 500 nm Ex/Em spectral acquisition in less than 1 s. Thus, this spectrophotometer is suitable in the study of metal-DOM complexation kinetics. The 3DEEM spectral data over time were collected and manipulated using Grams/32c software. Time-resolved 3DEEM fluorescence was determined for each run. Independent maximum Ex/Em wavelengths were determined in each spectrum. Ex/Em wavelengths were set at 350/450 nm to examine the changes in fluorescence intensity versus time. Pseudo-first-order plots of ln(FLt - FLe) versus time, where FLt is the fluorescence intensity at time t and FLe is the fluorescence intensity when pseudoequilibrium is achieved, were analyzed to determine objectively the possible number of straight-line components. Kinetic data were treated by the expression7,8
FL ) Ae-K1t + Be-K2t + ‚‚‚
(1)
where FL is the fluorescence intensity, K1, K2, ‚‚‚, are the observed pseudo-first-order rate constants for the reactions, and A, B, ‚‚‚, (15) Thurman, E. M.; Malcolm, R. L. Environ. Sci. Technol. 1981, 15, 463-466. (16) O’Driscoll, N. J.; Evans, R. D. Environ. Sci. Technol. 2000, 34, 4039-4045. (17) Wu, F. C.; Evans, R. D.; Dillon, P. J. Anal. Chim. Acta 2002, 464, 47-56.
Figure 2. Changes in fluorescence vs time in Cu(II) and CBFA complexation kinetics. (a) Fluorescence intensity at Ex/Em 350/400 nm, biphasic kinetic fit (line) and Rayleigh scattering (dashed line and circle) at Ex ) 540 nm. (b) Pseudo-first-order kinetics plot. (c) Excitation and emission wavelengths of fluorescence maximum.
are their constants. For the two straight-line components, A and B, K1 and K2 were obtained by SigmaPlot nonlinear regression program. RESULTS AND DISCUSSION Changes in Fluorescence Intensity during FA Complexation with Metals. Figures 2a, 3a, 4a, and 5a and 6a show changes in total fluorescence intensity after quickly mixing CBFA solution with several metal solutions (Cu2+, Ni2+, Co2+, Cd2+, Ca2+) using the stopped-flow apparatus. The fluorescence intensity rapidly decreased as the reaction of FA-metal complexation proceeded. Previous studies have demonstrated that the Rayleigh scattering can be used to monitor precipitate formation during metal titration as it is related to the number of particles in the solution.14 Figures 2a, 3a, 4a, 5a, and 6a show the Rayleigh scattering data collected simultaneously with fluorescence during the FA-metal complexation process. The constant Rayleigh scattering indicates that no coagulation or precipitation formation was occurring. Therefore, the fluorescence decrease was solely due to the metal quenching. This is consistent with a previous study, in which the binding of copper to fulvic acid was found to be fairly rapid.10 Although bivalent metals, e.g., Ni2+, Co2+, Cd2+, and Ca2+, were reported to be weak quenchers compared to Cu2+,18,19 their complexation with FA was similar to Cu2+ in that their reactions were virtually at equilibrium after ∼20-30 s. The reactions of the bivalent metals with FA followed pseudo-first-order kinetics. As shown in Figures 2b, 3b, 4b, 5b, and 6b, the plots for all metals studied consisted of two major linear segments with discernible slopes, indicating that there were two major kinetically distinguishable components on FA for each of those metals. This observation is in agreement (18) Saar, R. A.; Weber, J. H. Anal. Chem. 1980, 52, 2095-2100. (19) Cabaniss, S. E. Environ. Sci. Technol. 1992, 26, 1133-1139.
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Figure 5. Changes in fluorescence vs time in Cd(II) and CBFA complexation kinetics. (a) Fluorescence intensity at Ex/Em 350/400 nm, biphasic kinetic fit (line) and Rayleigh scattering (dashed line and circle) at Ex ) 540 nm. (b) Pseudo-first-order kinetics plot. (c) Excitation and emission wavelengths of fluorescence maximum. Figure 3. Changes in fluorescence vs time in Ni(II) and CBFA complexation kinetics. (a) Fluorescence intensity at Ex/Em 350/400 nm, biphasic kinetic fit (line) and Rayleigh scattering (dashed line and circle) at Ex ) 540 nm. (b) Pseudo-first-order kinetics plot. (c) Excitation and emission wavelengths of fluorescence maximum.
Figure 6. Changes in fluorescence vs time in Ca(II) and CBFA complexation kinetics. (a) Fluorescence intensity at Ex/Em 350/400 nm, biphasic kinetic fit (line) and Rayleigh scattering (dashed line and circle) at Ex ) 540 nm. (b) Pseudo-first-order kinetics plot. (c) Excitation and emission wavelengths of fluorescence maximum. Figure 4. Changes in fluorescence vs time in Co(II) and CBFA complexation kinetics. (a) Fluorescence intensity at Ex/Em 350/400 nm, biphasic kinetic fit (line) and Rayleigh scattering (dashed line and circle) at Ex ) 540 nm. (b) Pseudo-first-order kinetics plot. (c) Excitation and emission wavelengths of fluorescence maximum.
with a previous study of aluminum-FA interactions in which two general types of Al binding sites on FA have been postulated.8 This is also consistent with the results of Cabaniss,19 who reported that at least two fluorescent binding sites were present in fulvic acid for most of the metals examined at pH 7.5. There might exist at least three binding sites on fulvic acid as Kumke et al.,20 Kumke (20) Kumke, M. U.; Tiseanu, C.; Abbt-Braun, G.; Frimmel, F. H. J. Fluoresc. 1998, 8, 309-320.
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and Frimmel,21 McGown et al.,22 and Cook and Langford23 observed at least three lifetime components of fulvic acid of different origin, Cu- and Ni-fulvic acid complexation using timeresolved fluorescence measurements.20-23 The rate constants (K1 and K2) for the two major binding sites were calculated by nonlinear least-squares fitting of the (21) Kumke, M. U.; Frimmel, F. H. In Refractory Organic Substances in the Environment; Frimmel, F. H., Abbt-Braun, G., Heumann, K. G., Hock, B., Lu ¨ demann, H.-D., Spiteller, M., Eds.; John Wiley and Sons: New York, 2002; pp 264-281. (22) McGown, L. B.; Hemmingsen, S. L.; Shaver, J. M.; Geng, L. Appl. Spectrosc. 1995, 49, 60-66. (23) Cook, R. L.; Langford, C. H. Anal. Chem. 1995, 67, 174-180.
Table 1. Mean Observed Rate Constants (K1 and K2) for Reactions of Various Metals (Cu2+, Ni2+, Co2+, Cd2+, Ca2+) with CBFA, Their Half-Lives, and Relative Proportions of Two Kinetic Componentsa
Cu2+ Ni2+ Co2+ Cd2+ Ca2+ a
K1 (s-1) [half-lives (s)]
K2 (s-1) [half-lives (s)]
0.55 [1.26] 0.28 [2.48] 0.27 [2.57] 0.21 [3.30] 0.18 [3.85]
0.02 [34.7] 0.01 [69.3] 0.01 [69.3] 0.01 [69.3] 0.01 [69.3]
A (%)
B (%)
87.1
12.9
78.6
21.4
73.3
26.7
72.1
27.9
71.7
28.3
Values represent the average data of at least three runs.
ln(FLt - FLe) versus time plots (Table 1). The curves calculated using the predicted parameters fit well with the observed fluorescence data (Figures 2a, 3a, 4a, 5a, and 6a). Since FA is highly heterogeneous in nature, the calculated K1 and K2 values are, in reality, averages over kinetically distinguishable classes within the distribution of metal binding sites of FA. A and B in eq 1 were the fluorescence intensity, proportional to the initial fluorescence, of the two kinetic components of FA. As seen in Table 1, the K1 value, for the fast-reacting binding site, was in the same order of magnitude, ranging from 0.18 to 0.55 s-1 with the constant following the order, Cu2+ > Ni2+ > Co2+ > Cd2+ > Ca2+. This is in accordance with the Irving-Williams series for the binding strength of bivalent metal complexes of the first transition series with a given ligand17,24,25 and with proteins.26 The fast binding site was responsible for 71-87% of the total fluorescence decrease, with the remainder associated with the slow binding site. The relative proportions of the fast binding site (Table 1) were Cu2+ > Ni2+ > Co2+ > Cd2+ > Ca2+, which again agrees with the Irving-Williams series. The K2 values for the slow reacting binding site were similar for all metals at 0.01-0.02 s-1. As shown in Table 1, the half-lives of the fast reacting site were between 1.26 and 3.85 s, while those of the slow reacting site were 34.7-69.3 s, over 1 order of magnitude slower than those of the fast site. Previous studies showed that the kinetics of metal-FA complexation exhibited concentration, temperature, and pH dependence,8 as well as a molecular size dependence.10 This study demonstrates the correlation between the kinetics and the binding strength of the metal involved. The stronger the affinity of the metal, the greater proportion of strong and fast reacting FA binding sites involved in the complexation process. This finding indicates that metal affinity affects not only thermodynamic equilibrium but also its kinetic reaction process. (24) Irving, H.; Williams, R. J. P. J. Chem. Soc. 1953, 3, 3192-3196. (25) Winzerling, J. J.; Berna, P.; Porath, J. Methods 1992, 4, 4-10. (26) Sidenius, U.; Farver, O.; Jones, O.; Gammelgaad, B. J. Chromatogr., A 1999, 735, 85-91. (27) Ropson, I. J.; Dalessio, P. M. Biochemistry 1997, 36, 8594-8601.
Changes in Fluorescence Spectral Patterns during the FA-Metal Complexation Process. To determine possible changes in fluorescence spectra during the metal-FA complexation process, differential spectra were obtained by subtracting the spectra when pseudoequilibrium was reached. There was a single major fluorescence maximum in the differential spectra (data not shown). Figures 2c, 3c, 4c, 5c, and 6c show changes in both Ex and Em wavelengths of the fluorescence maximums in the differential spectra versus time. There were two major phases in the 30 s of reaction time for all metals studied. The first phase occurred within 4-5 s, in which the strong binding site reacted, with the second phase following between 5 and 30 s. In each phase, both Ex and Em wavelengths gradually increased with reaction time. At present, little is known about structural changes during FAmetal complexation kinetics since previously these reactions were monitored as total intensity changes. This gives no information regarding the structural nature of FA-metal complexation. There may be cases where a structural change has occurred, as marked by changes in the Ex and Em wavelengths of fluorescence maximums. In terms of both Ex and Em wavelengths, the observed two kinetic phases clearly suggest that a structural change occurred during the complexation process. This implies two major types of binding sites on FA. Metal ions may react initially with the fast reacting and strong binding site on FA; after the fast binding site is fully occupied, the slow reacting and weak binding site starts to bind with the metal ions. In each phase, the red-shifted Ex and Em spectra suggest a structural transition of the FA-metal complex with time. Possible structural changes may include conformational changes or changes in rigidness as the complexing process intensifies. To the best of our knowledge, this is the first report of full excitation and emission fluorescence spectral kinetics during FA-metal complexation. The deviation or variation from Figures 2b,c, 3b,c, 4b,c, 5b,c, and 6b,c suggests more than two binding sites on FA and their differences for various metals, as suggested by Cook and Langford, McGown et al., Kumke et al., and Kumke and Frimmel.20-23 However, it is interesting to note in this study that, in terms of the kinetic time, both the two straight linear portions of the pseudo-first-order kinetic plots and two step phases of maximum Ex and Em wavelengths were consistent, as shown in Figures 2b,c, 3b,c, 4b,c, 5b,c, and 6b,c. This indicates that two major binding sites may be a common feature of FA and may represent real structural changes that occur in the FA-metal complexation. The result provides further strong support for the biphasic kinetic analyses widely used in previous reports.7,8,27 Synchronous quenching spectra were reported to provide a useful tool for directly examining binding sites on FA.19 This study demonstrates that the use of full fluorescence spectra can provide additional information in the formulation of chemically reasonable models for metal binding by FA or DOM and in obtaining additional structural information on the complexation process. Received for review January 2, 2003. Accepted September 30, 2003. AC030005P
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